1. Field of the Invention
The invention is in the field of metallic stains for biopolymers such as proteins, polypeptides and nucleic acid molecules when fixed in synthetic matrixes.
2. Related Art
Gel electrophoresis is a commonly used analytical technique in biochemistry and related fields of study for the separation of nucleic acids, polypeptides, proteins and oligosaccharides. A sample of interest is placed in a matrix and exposed to an electric field which causes various components to migrate and separate into distinct bands dependent upon the molecular weight, charge, and other physical properties of the molecules. After electrophoresis has ended, the pattern of migration is not typically decipherable because the majority of molecules have no integral choromophores or fluorophores by which they may be visualized. Numerous methods have been developed to visualize the exact locations of the molecules of interest within a matrix while leaving the blank areas of the matrix virtually unstained. These include the Coomassie Brillant Blue dyes, ethidium bromide and Ponceau S stains. Silver staining was developed to increase the sensitivity over that achieved with these dyes. One of the earliest and widely used silver staining technique was reported by Merril et al., Meth. Enzymol. 96:230 (1983). In this report, an electrophoretic matrix, specifically polyacrylamide, is immersed in either an acid or an acid/alcohol solution for about one hour to fix the proteins within the matrix. The matrix is then washed typically for about thirty minutes. The matrix is then soaked for about five minutes in a dichromic acid solution to oxidize the protein. Next, the gels are soaked in a silver nitrate solution for twenty minutes and then rinsed with a sodium carbonate/formaldehyde buffer to reduce silver ions bound to proteins and nucleic acids. A silver pattern is allowed to develop and then stopped by the addition of acetic acid. The pattern is then analyzed either by direct visualization or by instrumental techniques.
The method of Merril et al. was simplified by Oakley et al., Anal. Biochem.105:361 (1980). Electrophoresed gels were treated with unbuffered glutaraldehyde to cross-link proteins. Following rinsing, the gels were treated with a silver nitrate/ammonium hydroxide/sodium hydroxide solution. Finally, a solution of formaldehyde in citric acid is used to reduce silver ion to silver and visualize the bands within the gel matrix.
Glutaraldehyde has been employed by many laboratories to lower the limit of detection of proteins by silver staining (Rabilloud, T., Electrophoresis 11:785–794 (1990); Rabilloud, T., Cell. Mol. Biol. 40:57–75(1994)). The exact mechanism by which this compound enhances sensitivity has not been evaluated extensively, but it is thought to function by first binding to free amino groups in the protein to form a Schiff base leaving a free aldehyde function. Free amino groups are found at the N-terminus of the protein and also on the side chain of lysines and arginines. This aldehyde may reduce silver in the protein zone leading to an initiation of silver metal deposition. Silver metal is thought to catalyze further reduction of silver ion to silver so the net result is to increase the rate of silver deposition adjacent to the protein. An example of such a product employing glutaraldehyde sensitization is the SILVERXPRESS silver stain sold by Invitrogen Corporation, Carlsbad, Calif.
Use of glutaraldehyde, though it improves sensitivity of the stain to low levels of proteins, has an undesirable effect when further analysis of the gel by mass spectrometry is desired. Scheler et al., Electrophoresis 19:918–927 (1998). A widely used technique for the analysis of proteins by mass spectrometry is to cleave the protein into fragments with the enzyme trypsin. The fragments of a protein created by tryptic cleavage are readily predicted because the enzyme preferentially cleaves the amide bond immediately after a basic amino acid such as lysine or arginine. If the amino group of lysine or arginine is bound in a Schiff base by glutaraldehyde, it is no longer a cleavage site for trypsin. In addition, when an initial complex is formed between the glutaraldehyde molecule and the protein, the remaining free aldehyde group is available to condense with other amino groups thereby producing crosslinked proteins. This property is what makes glutaraldehyde such an effective fixative for histological applications. However by crosslinking peptides and proteins containing amino groups in a random fashion, not only are the potential trypsin cleavage sites blocked, crosslinked peptides and peptides are randomly created whose molecular weight may not be predicted from an analysis of the protein primary structure. As a result, there is a reduced abundance of many fragments whose molecular weight would be diagnostic for the identity of the protein. Although other enzymes can be used to fragment the protein, the specificity of trypsin makes it a favored choice.
U.S. Pat. No. 4,405,720 discloses a silver staining method for polypeptides in gels comprising photo-reversing the polypeptide-gel by treatment with an oxidizing reagent, forming a latent stain image by treating the polypeptide-gel with a reduceable metal salt termed a photosensitive salt, and developing the stain image by treating the polypeptide gel with a reducing agent. Examples of such photosensitive salts include salts of silver, gold, platinum, palladium and/or iridium.
U.S. Pat. No. 4,468,466 discloses a silver staining method comprising treatment with the reducing agent dithiothreitol followed by treatment with a silver salt and actuating radiation. According to this patent, dithiothreitol acts as a reducing agent to effect photoreversal and avoid silver staining of non-proteins.
U.S. Pat. No. 4,703,016 discloses a silver staining procedure for proteins and DNA. The process comprises fixing a protein on a membrane in cupric acetate solution; contacting the membrane with a solution comprising acetic acid, sodium chloride, and citric acid; contacting the membrane with a solution comprising acetic acid and silver nitrate and irradiating with a light source; contacting the membrane with a solution comprising acetic acid, sodium chloride and citric acid; transferring the membrane back to the silver nitrate solution and irradiating the membrane; developing the image by transferring to a solution comprising hydroquinone and formaldehyde; washing with water, contacting with sodium thiosulfate; and then washing with water.
U.S. Pat. No. 4,575,452 discloses methods of detection of proteins and nucleic acids in a matrix comprising fixing the proteins and nucleic acids in the matrix with aromatic sulfonic acid compounds having tertiary amines and N,N′-di-(9-acridyl)-diaminoalkylene compounds. Particular examples of such compounds include 4,4′-[1,4-phenylenebis(2,5-oxazolediyl)]-bisbenzene-sulfonic acid, 4,4′-[1,4-phenylenebis(4-methyl-2,5-oxazolediyl)]bisbenzene-sulfonic acid, 2,2′-(2,5-thiophenediyl)bis[5-(1,1-dimethylethyl)-7-benzoxazole-sulfonic acid, N,N,N-trimethyl-2-phenyl-5-(4-sulfophenyl)-4-oxazolemethanamonium hydroxide, 2,2′-(1,4-phenylene)bis[N,N,N-trimethyl-5-(4-sulfophenyl)]-4-oxazolemethanamonium hydroxide, and N,N′-di-(9-acridyl)-1,6-diaminohexane.